Optimization of bacteriocin release protein (BRP)-mediated protein release by Escherichia coli: random mutagenesis of the pCloDF13-derived BRP gene to uncouple lethality and quasi-lysis from protein release.
Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli periplasm into the culture medium. However, high-level expression of BRP causes apparent lysis of the host cells in liquid cultures (quasi-lysis) and inhibition of growth on broth agar plates (lethality). To optimize BRP-mediated protein release, the pCloDF13 BRP gene was subjected to random mutagenesis by using PCR techniques. Mutated BRPs with a strongly reduced capacity to cause growth inhibition on broth agar plates were selected, analyzed by nucleotide sequencing, and further characterized by performing growth and release experiments in liquid cultures. A subset of these BRP derivatives did not cause quasi-lysis and had only a small effect on growth but still functioned in the release of the periplasmic protein beta-lactamase and the periplasmic K88 molecular chaperone FaeE and in the release of the bacteriocin cloacin DF13 into the culture medium. These BRP derivatives can be more efficiently used for extracellular production of proteins by E. coli than can the original BRP.
van der Wal
F J
FJ
Department of Molecular Microbiology, BioCentrum Amsterdam Faculty of Biology, Vrije Universiteit, The Netherlands.
Koningstein
G
G
ten Hagen
C M
CM
Oudega
B
B
Luirink
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D001430:Bacteriocins / Q000378:metabolism; D001483:Base Sequence; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D001618:beta-Lactamases / Q000378:metabolism
D001426:Bacterial Proteins; D001430:Bacteriocins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; C052368:cloacin DF13-encoded bacteriocin release protein, E coli; C070012:faeE protein, E coli; D001618:beta-Lactamases